Project Summary/Abstract Managing tumor recurrence and spread is a major challenge in breast cancer. This scenario is further aggravated for women with diabetes as numerous epidemiological studies show that women with type 2 diabetes are at significantly greater risk of developing, relapsing with, and dying from breast cancer compared to women who are not diabetic. Studies show that hyperinsulinemia associated with type 2 diabetes is a significant contributing factor for the mortality seen in breast cancer patients, suggesting an important need for effective therapies that inhibit tumor cell proliferation under hyperinsulinemic conditions. Our early studies showed that cdk4 knock- out mice fail to develop breast cancers driven by the ERBB2 or RAS oncogenes suggesting that CDK4/6 inhibitors may be effective therapeutic agents for certain breast cancers. The approval of CDK4/6 inhibitors as breast cancer therapeutics validated this theory. Another kinase which plays a critical role in insulin signaling and cancer progression is NUAK1/ARK5 which belongs to the AMPK gene family which regulate metabolism. The normal physiological role played by NUAK1/ARK5 in the whole organism was studied using muscle-specific knock-out mice which showed that NUAK1 controls glucose metabolism through regulation of the insulin signaling. Thus, when these knock-out mice were fed a high fat diet, they exhibited a lower fasting blood glucose level, greater glucose tolerance, higher insulin sensitivity, and higher concentrations of muscle glycogen compared to control mice suggesting that inhibition of ARK5/NUAK1 can overcome the effects of hyperglycemia. Interestingly, ARK5 was originally identified as a metastasis gene and its over-expression has been shown to promote metastasis of several tumor types. To achieve the goal of inhibiting breast tumor cell growth and metabolism, we developed a potent dual inhibitor of CDK4 and ARK5 (ON123300), which was a very effective inhibitor of breast tumor growth. In addition, this compound has shown a profound effect on high sugar diet-induced tumor development and metastasis in a Drosophila model system. In this application, we propose to extend these studies to mouse models of hyperinsulinemia (MKR mice), developed by Dr. LeRoith and PDX models of Triple Negative Breast cancer (TNBCs) developed by Dr. Irie. The aims are: (1) To test the effects of ON123300 on the growth and metastasis of mammary tumors in MKR mice which exhibit hyperinsulinemic, pre-diabetic phenotype; (2) To examine the effects of hyperinsuminemia on the growth and metastasis of PDX breast tumors and utilize the models with the highest relative levels of ARK5 to examine the therapeutic value of ON123300; and (3) To use the newly developed Multiplexed-kinase Inhibitor Beads (MIB) and ?Cancer Toolkit gain-of-function? (CTK) technologies to determine whether breast cancer cells develop resistance to ON123300 upon prolonged exposure and to determine the nature of signaling pathways that might be the root cause of such resistance.